Biotin (vitamin H), an essential coenzyme required by all forms of life, is only synthesized by plants, most bacteria and some fungi. In living cells, a few metabolic enzymes are naturally biotinylated through post-translational modification that is carried out by an intracellular enzyme, biotin protein ligase (BPL), also known as holocarboxylase synthetase [EC 6.3.4.10]. BPL catalyzes the formation of an amide linkage between the carboxyl group of biotin and the ε-amino group of a specific lysine residue of the substrate protein in a two-step reaction ([Cronan, 1990] and [Wood and Barden, 1997]). The non-covalent interaction between avidin/streptavidin and biotin represents one of the strongest and most specific interactions amongst biological molecules (KD=10−14 to 10−15 M). This property has been exploited by researchers who have attached biotin ‘tags’ to proteins for easy detection, labeling, immobilization and purification ([Cull and Schatz, 2000], [Kumar and Snyder, 2002], [de Boer et al., 2003], [Kojima et al., 2006] and [Krepkiy et al., 2006]). Biotin labeling has also been applied to drug targeting ([Ohno et al., 1996] and [Asai et al., 2005]) and viral gene therapy vector-targeting strategies ([Smith et al., 1999], [Parrott et al., 2003], [Campos and Barry, 2004] and [Arnold et al., 2006]).
Biotin labeling of drugs, proteins or virus has traditionally been performed in vitro by chemical methods, where an activated biotin derivative is conjugated to protein surface residues (commonly lysines) or carbohydrate moieties ([Bayer and Wilchek, 1990], [Diamandis and Christopoulos, 1991], [Ohno et al., 1996] and [Smith et al., 1999]). However, these methods result in random and heterogeneous modification, which can lead to the inactivation of biological function and cross-linking or aggregation after mixing with streptavidin or avidin. Antibody biotinylation by chemical methods generally leads to the preparation of heterogeneous conjugates. Furthermore, biotinylation of the residues in the binding site of antibodies can alter their binding properties (Saviranta et al., 1998) and result in loss of affinity.
An alternative approach to chemical methods was first demonstrated by Cronan (1990). Fusion of the biotin attachment sites of proteins from four different species to the carboxyl terminus of β-galactosidase enabled biotinylation in Escherichia coli by endogenous biotin ligase. The functional interaction between biotin ligases and their protein substrates shows a very high degree of conservation throughout evolution, since biotinylation occurs even with enzymes and substrates from widely divergent species (Chapman-Smith and Cronan, 1999). The most studied endogenous biotinylated protein is the 1.3S subunit of the transcarboxylase domain of Propionibacterium shermanii (PSTCD), which is structurally very similar to that of E. coli acetyl-CoA carboxylase (Reddy et al., 1998). By fusing the biotin acceptor peptide domain of PSTCD to the target protein, it was demonstrated that biotinylation could occur in bacterial, yeast, insect and mammalian cells ([Smith et al., 1999], [Parrott and Barry, 2001] and [Verhaegen and Christopoulos, 2002]). A recent in vivo imaging study showed that tumor cells expressing PSTCD tagged surface receptor protein was detected using a variety of imaging agents coupled to streptavidin (Tannous et al., 2006). Biotinylation can occur either by cellular endogenous protein-biotin ligase or by the coexpression of an exogenous biotin ligase, in most cases that of bacterial BirA enzyme (Tsao et al., 1996).
Smaller peptide tags (<23 aa) identified by peptide libraries were also found to be biotinylated in vitro with kinetics comparable to those of natural biotin acceptor sequence (Schatz, 1993). A 15 residue peptide (GLNDIFEAQKIEWHE (SEQ ID NO:1), Biotin AviTag™) (Beckett et al., 1999) with 100% biotinylation efficiency was used for specific biotinylation of fusion protein in E. coli, insect and mammalian cells ([Smith et al., 1998], [Wu et al., 2002], [de Boer et al., 2003], [Viens et al., 2004], [Yang et al., 2004], [Warren et al., 2005] and [Tirat et al., 2006]). Utilizing this small peptide in vivo biotinylation has also been performed on the surface of yeast (Parthasarathy et al., 2005).
Antibodies can be engineered into a variety of formats that retain binding specificity and exhibit optimal properties for in vitro or in vivo applications. Single-chain antibody fragments (scFvs), produced by genetically fusing variable light (VL) and heavy (VH) chain domains of a parental antibody through a peptide linker, represent the smallest functional unit (25-30 kDa) that still retains the capacity to bind antigen. Production of single-chain antibody scFv dimers (also known as diabodies, 55 kDa) can be forced by shortening the peptide linker, which in turn enhances the binding activity (Holliger et al., 1993).
Despite recent advances in the chemical and metabolic methods of producing biotinylated polypeptides, there remains a need in the art for highly specific and highly efficient methods of producing large quantities of biotinylated polypeptides for applications such as medical diagnostics and pharmaceutical administration. The present invention fulfills these and other need by providing novel methods, cell lines, systems, and kits for efficient metabolic biotinylation of secreted polypeptides.